The packaging of DNA into chromatin restricts the accessibility of DNA to factors involved in fundamental cellular processes such as DNA replication and transcription. The repeating organizing unit of chromatin is the nucleosome. Each nucleosome consists of a core built of two copies of histones H2A, H2B, H3, and H4, around which the DNA is tightly wrapped and bound by electrostatic interactions. Consistent with the repressive effects of chromatin on gene expression, gene activation is often accompanied by nucleosomal rearrangements. Such local or extended structural changes in chromatin are achieved by ATP-driven chromatin remodeling complexes and by posttranslational acetylation, methylation or phosphorylation of histones.
The most abundant and best-studied posttranslational modification of chromatin is the reversible acetylation of lysines in the amino-terminal tails of the four core histones. Transcriptionally silenced regions, such as heterochromatin and the inactivated mammalian X chromosome, are associated with hypoacetylated histones. In contrast, transcriptionally active domains in euchromatin are often associated with histone hyperacetylation. Localized changes in histone acetylation levels near the transcriptional start site of certain genes are linked to gene activation or repression.
The causal link between histone acetylation and transcriptional regulation is dramatically illustrated by the identification and characterization of transcriptional regulators containing histone acetyltransferase (HAT) or deacetylase (HDAC) activities. HDAC proteins are classified in three distinct families. Class I HDACs (HDAC1, 2, 3, and 8) are derived from the yeast transcriptional regulator RPD3. Class II HDACs (HDAC4, 5, 6, and 7) are similar to HDA1, another deacetylase in yeast. Class III HDACs are related to the yeast silencing protein SIR2 and are dependent on NAD for enzymatic activity. In contrast to the relative compact proteins of class I, class II HDACs (HDAC4, 5, and 7) possess two distinct domains. The carboxyl-terminal domain of HDAC4 has catalytic activity in vivo whereas the amino-terminal domain exerts autonomous repressor activity by an unknown mechanism.
Class I HDACs are expressed in most cell types; class II HDACs are expressed in a tissue-specific manner and have been implicated in the regulation of muscle differentiation. HDACs 4 and 5 bind to transcription factors of the MEF2 family via their amino-terminal domain. This binding is regulated by calcium through calmodulin and calmodulin-dependent kinases. Both HDAC4 and HDAC5 shuttle in and out of the cell nucleus in a regulated manner controlled by phosphorylation and interaction with 14-3-3 proteins. Class II HDACs 4, 5 and 7 interact with SMRT, N-CoR, and BCoR, an additional corepressor that mediates repression by BCL-6.
HDACs are part of high-molecular-mass corepressor complexes. These complexes are recruited to specific promoters via their interactions with sequence-specific DNA binding proteins, including the nuclear hormone receptors, the E-box binding factors, and the methylcytosine-binding protein MeCP2. Class I HDAC1 and HDAC2 are found in SIN3 and NURD/Mi2 complexes. In contrast, HDAC3 is associated with the corepressors SMRT and N-CoR in a complex that mediates transcriptional repression by the thyroid hormone receptor and the oncoprotein v-ErbA.
Mutational analysis of RPD3 and HDAC1 indicates that enzymatic activity is essential for the transcriptional regulatory functions of these proteins in vivo. However, with few exceptions, it has been remarkably difficult to obtain recombinant HDAC proteins demonstrating enzymatic activity in vitro. Immunoprecipitation experiments have been required to study and characterize the enzymatic activity of most HDACs. In addition, biochemical fractionation of mammalian cell extracts has indicated that several additional proteins beside HDAC1 and HDAC2 might be necessary to build an enzymatically active HDAC1/HDAC2 core complex. The limitations observed in the expression of recombinant HDAC activity could therefore be the consequence of missing essential cofactors.